†Contributed equally to this work *Corresponding author (email: [email protected])
• RESEARCH PAPER • February 2012 Vol.55 No.2: 172–180
doi: 10.1007/s11427-012-4276-0
Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa
LUO YuShuang†, KOU XiaoXiao†, DING XueZhi, HU ShengBiao, TANG Ying, LI WenPing, HUANG Fan, YANG Qi, CHEN HanNa & XIA LiQiu*
State Key Laboratory of Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, Changsha 410081, China
Received November 18, 2011; accepted December 5, 2011
To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopoly-spora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was fur-ther confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vit-reoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxy-gen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.
Citation: Luo Y S, Kou X X, Ding X Z, et al. Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccha-ropolyspora spinosa. Sci China Life Sci, 2012, 55: 172–180, doi: 10.1007/s11427-012-4276-0
The Vitreoscilla hemoglobin gene (vgb) is located in the chromosomes of Vitreoscilla stercoraria, a strict aerobic gram-negative filamentous bacterium. Upon gene transcrip-tion, vgb is induced to synthesize a soluble heme protein called Vitreoscilla hemoglobin (VHb) in relatively large quantities when the strain is grown under hypoxic condi-tions, thereby satisfying the demand for dissolved oxygen. Originally, VHb was recognized as a “cytochrome o” with terminal oxidase activity [1]. Spectroscopic analysis re-vealed that VHb shared similar oxygen binding kinetics with myoglobin and hemoglobin, and its transition among three states (reduced form, oxidized form and oxygenated
hemoglobin) depended on the concentration of dissolved oxygen present [2]. Ultramicroscopic structure analysis of cells containing VHb indicated that it is located in the cyto-plasm and is concentrated near the periphery of the cyto-solic face of the cell membrane [3]. Early research on the mechanism of VHb action demonstrated that under hypoxic growth conditions it could capture oxygen and facilitate its transfer to the respiratory membranes by interacting with terminal oxidase, thereby enabling cells to adapt to the hy-poxic environment [4]. Recent research on the metabolic regulation and proteomics of VHb has shown it also has peroxidase activity [5]. Its expression can regulate cells physiological function, eliminate unfavorable effects of by-products such as organic acid production during fermen-tation [6,7] and regulate the energy and metabolism of cen-
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tral intermediates [8,9]. VHb has become a versatile tool in high-density fermentation of micro-organisms susceptible to oxygen supply [10,11]. Recent studies of VHb applications include: (i) promoting the biosyntheses of biochemical pre-cursors (such as Dihydroxyacetone) [1215]; (ii) improving bioremediation (improving desulfidations in Rhodococcus erythropolis) [16]; (iii) enhancing the production of im-portant enzymes (such as D-amino acid oxidase) [17,18]; (iv) improving the biosyntheses of antibiotics and biopesti-cides [19,20], and (v) promoting the production of biopol-ymer materials (such as poly-γ-glutamic acid) [21,22].
Spinosyns are secondary metabolites produced from the aerobic fermentation of S. spinosa. The spinosyns are members of a new class of polyketide-derived macrolides effective against a broad range of insect pests. Originally, they were designated as A83543 factor [23]. The most ac-tive and abundant components of the spinosyn family of compounds are spinosyn A and spinosyn D (spinosad). Pre-vious research showed that spinosad has rapid contact and ingestion toxicity to insects. It can kill susceptible insects such as Lepidoptera and Thysanoptera by causing rapid excitation of the insect nervous system, probably through binding and interacting with the nicotin acetylcholine and -amino butyric acid receptors [24]. Importantly, this new class of insecticides showed little or no effect on a broad range of non-target insects and mammals [25]. To date, no other class of product has been found to affect the insect nervous system with the same mode of action and no cross-resistance to spinosad has been reported [26]. Spi-nosad is considered to be a promising biopesticide world-wide and has been widely used in agriculture as a potent insect control agent and animal health product because of its high efficiency against target insects and environ-ment-friendly characteristics.
However, spinosad levels in wild type S. spinosa are low, and therefore considerable interest has been shown in iden-tifying ways to improve its yield. Previously reported methods to improve S. spinosa spinosad levels include clas-sic mutagenesis and rational selection [27], protoplast re-generation [28], intergeneric protoplast fusion [29], and duplication of the genes involved in spinosad biosynthesis [30]. S. spinosa has a filamentous growth and used fatty acid as precursor for spinosad biosynthesis, certain amount oil need to be added during fermentation. This results in a viscous culture and a consequent shortage of oxygen, which is one of the limiting thresholds of spinosad production. In this study, we attempted to improve spinosad biosynthesis through enhancing the oxygen supply during high-density fermentation by expression of VHb in S. spinosa.
1 Materials and methods
1.1 Bacterial strains, plasmids and primers
Bacterial strains, plasmids and primers used in this study are
listed in Table 1.
1.2 Media and cultivation conditions
All E. coli strains were grown in Luria-Bertani (LB) medium or TY liquid medium [31]. Single clone or 1% spawn preservation was inoculated into 10 mL LB medium sup-plemented with corresponding antibiotics and incubated at 37°C with shaking at 200 r min1 overnight. Seed medium CSM (tryptic soy broth 30 g L1, yeast extract 3 g L1, glu-cose 5 g L1, maltose 4 g L1) was used for the activation of S. spinosa. 0.5 mL of the bacteria stored in glycerol (stored at –80°C) was inoculated into 250 mL-flasks containing 20 mL CSM. Five glass beads (~5 mm) were added to each flask and incubated at 30°C with shaking at 300 r min1 for 48 h. R6 and BHI (brain heart infusion) solid medium [32] was used for conjugation between E. coli and S. spinosa. The fermentation medium and cultivation conditions of S. spinosa were previously described [28].
1.3 Treatment with enzymes, reagents and antibiotics
LA Taq DNA polymerase, restriction endonuclease, T4 DNA ligase, and agarose gel DNA extraction kits were purchased from Takara Biotechnology Co., Ltd. (Dalian). PCR primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai). Tryptone and yeast extract were purchased from Oxiod. TSB and BHI were purchased from Difco. Digoxin labeling and detection kit were purchased from Roche. Oth-er reagents (analytical grade or chromatographic grade) were purchased from China National Medicines Corpora-tion Ltd. Apramycin (Apr, 50 mg L1), ampicillin (Amp, 50 mg L1), chloramphenicol (Cm, 25 mg L1), tetracycline (Tet, 25 mg L1), kanamycin (Kan, 50 mg L1), and nalidix-ic acid (NA, 25 mg L1) antibiotics were purchased from Sigma.
1.4 Basic operation of gene technology
Preparation of E. coli competent cells, plasmid extraction, restriction enzyme digestion, ligation, transformation, PCR, electrophoresis and Southern blotting were performed ac-cording to Molecular Cloning: A Laboratory Manual [31]. Genomic DNA extraction of S. erythraea and S. spinosa was performed according to Practical Streptomyces Genet-ics [33]. All PCR products were ligated into pMD18-T vec-tor and analyzed by restriction enzyme digestion. Positive clones were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).
1.5 Construction of the integrating vector pSET152- EVHB
The promoter of erythromycin resistance gene (PermE) was
174 Luo Y S, et al. Sci China Life Sci February (2012) Vol.55 No.2
amplified from the genomic DNA of S. erythraea. The pri-mers, P1 and P2, were designed according to the sequences published in GenBank (gi: 152688). The EcoR I restriction site was introduced into the 5′ end of P1 and the mutation site of CAGCAC was introduced into P2. The PCR was carried out in a volume of 50 μL containing 16.8 μL ddH2O, 25 μL 2× GC buffer, 2 μL 10 mmol L1 dNTP, 2 μL P1, 2 μL P2, 2 μL DNA template and 0.2 μL LA PCR Taq DNA polymerase. The PCR was conducted as follows: denatura-tion at 94°C for 2 min followed by 28 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and terminated by incuba-tion at 72°C for 5 min. The open reading frame (ORF) of vgb was amplified from the plasmid pRK404-VHb. The primers, P3 and P4, were designed according to the se-quences published in GenBank (gi: 155317). The Xba I re-striction site was introduced into the 5′ end of P4 and the mutation site of GTGCTG was introduced into P3. The PCR was carried out in a volume of 50 μL containing 35.5 μL ddH2O, 5 μL 10× buffer, 3 μL 2.5 mmol L1 dNTP, 2 μL P3, 2 μL P4, 2 μL template and 0.5 μL Cas Taq polymerase. The PCR was conducted as follows: denaturation at 94°C for 2 min followed by 28 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and terminated by incubation at 72°C for 5 min. The ORF of vgb was placed under control of the PermE promoter by splicing using an overlapping ex-tension PCR (SOE-PCR), using P1 and P4 as primers and the mixture of PermE and vgb PCR product as a template. The sequence of the PermE-vgb cassette was confirmed by TA-colony and DNA sequencing. The integrating vector pSET152EVHB was constructed by placing the PermE-vgb cassette between the EcoR I and Xba I sites in pSET152.
1.6 Construction of recombinant S. spinosa S078-1101
The plasmid pSET152EVHB was transformed into E. coli ET12567 (pUZ8002) competent cells and the transformants were selected for Apr, Cm and Kan resistance. The positive E. coli ET12567 (pUZ8002, pSET152EVHB) transformants were confirmed by enzyme digestion after plasmid extraction. E. coli-S. spinosa conjugation was conducted as previously described [32,34] with minor modifications. The recipient stock of S. spinosa SP06081 was cultured in CSM broth, and after 48 h growth, mycelia were ground with a glass homogenizer. They were then transferred with 10% of the inoculation into TSB broth in an Erlenmeyer flask and placed on a rocker (300 r min1, 30°C) for 16 h. After 16 h the culture was transferred into TSB broth with 25% of the inoculation, and after 6 h growth, cells were collected and resuspended in 2 mL TSB. Meanwhile, the overnight- cultured donor bacterium E. coli ET12567 (pUZ8002, pSET152EVHB) was washed twice with TSB to remove residual antibiotics and resuspended in 5 mL TSB. The do-nor-recipient (E. coli-S. spinosa) was mixed in the ratio 1:3 (250 μL:750 μL) and added to a sterile centrifuge tube. Then 1 mL of the mixture was plated on R6 medium and
placed in an ultra clean cabinet for 30 min to be fully ab-sorbed. The conjunction plates were inverted and incubated at 30°C for 16 h, then overlaid with Apr and NA to a final concentration of 50 and 25 μg mL1 respectively. A single colony on the conjunction plates was selected after 10–14 d culture and inoculated on a BHI plate containing Apr and NA. After 3–4 d growth, partial mycelia of the colonies were transferred into 15 mL TSB containing 50 μg mL1 Apr and 25 μg mL1 NA in a 250 mL Erlenmeyer flask, and stirred for 3–4 d at 30°C. The positive conjugants, named S. spinosa S078-1101, were confirmed by PCR amplification of the Apr and vgb gene, and then preserved at –80°C for subsequent analysis.
1.7 Analysis methods
1.7.1 Determination of VHb biological activity in recom-binant S. spinosa
Carbon monoxide (CO) differential spectrum assay was used to detect VHb biological activity [35]. The mycelia were collected after 5 d growth in fermentation medium, washed twice with pre-cooling buffer (10 mmol Tris-HCl, pH 7.0, 60 mmol NH4Cl, 10 mmol CH3COOMg, 1 mmol DTT, 1 mmol phenylmethanesulfonyl fluoride), centrifuged at 4°C and resuspended in 10 mL buffer. Cells were then placed into an ice water mixture and broken into pieces by means of sonication three times (30 cycles at 100 W power, ultrasound for 5 s with a 15 s interval). After centrifugation at 3000 r min1 below 4°C, 0.4 g sodium dithionite was added to the supernatant, until a saturated concentration was reached. Then the supernatant was divided into two after being reduced at room temperature for 20–30 min: one part was directly poured into a reference cell, and the other was exposed to CO for 3 min and placed without shaking in the dark for 10–15 min, before being poured into a detector cell. The spectral scan was performed between 400 and 500 nm.
1.7.2 Influence of VHb gene expression on the growth of S. spinosa
The A600 value was used to analyze the growth of S. spinosa in CSM medium. The bacterial nucleic acid value was ap-plied to determine indirectly the biomass of S. spinosa in the fermentation medium [36]. Each experiment was re-peated three times.
1.7.3 Comparative analysis of spinosad yield between the SP06081 and S078-1101 strains under different dissolved oxygen conditions
Fermentation for spinosad production was run for 9 d in a humidified rotary shaker incubator (NBS INNOVO 4900, USA) at 30C and 80 % relative humidity. The ÄKTA Puri-fier10 (GE Healthcare, USA) high-pressure liquid chroma-tography (HPLC) system was used for the analysis of spi-nosad yield. 1 mL fermentation broth was extracted with 1
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mL of methanol for 8–10 h at 30C. Methanol extracts were collected by centrifugation at 10000 r min1 for 15 min. After filtration through 0.22 µm Millipore filters, the super-natants (10 μL) were loaded onto a C18 column (AQ12S05-1546WT, 150 mm×4.6 mm ID, S-5 μm, 12 nm) then eluted with methanol/acetonitrile/2% aqueous ammo-nium acetate (v/v/v=45:45:10) at 1.5 mL min1. The detec-tion wavelength was 250 nm. A single point external stand-ard method was used for the quantitative analysis of spi-nosad in HP/C–900 chromatography working station. Three biological replicates were performed for each group exper-iment, and the data obtained were analyzed for ANOVA using SPSS 15.0 software package and the results were ex-pressed as the mean±SD.
1.8 Genetic stability analysis of S. spinosa S078-1101
The stock S078-1101 strain stored at –80C was cultured in CSM broth without antibiotics for 48 h, and then the cells were plated on R6 medium without antibiotics after gradient dilution and incubated for 5–7 d at 30C. One hundred strains were selected and inoculated on R6 plates containing Apr (50 mg L1), and the number of colonies with antibiotic resistance was counted. Then 20 strains were randomly se-lected from these resistant clones and transferred into CSM broth, and PCR amplification of the vgb gene from the strain cultures was performed after 48 h growth. Meanwhile, the 48 h old cultures were transferred into fermentation me-dium without antibiotics, and the spinosad yields were ana-lyzed by HPLC after 9 d fermentation. After five passages by this means, the genetic stability of the recombinant strain was investigated by comparative analysis of the spinosad yields between generations, taking the spinosad yield of the first generation of the recombinant strain as 100%.
2 Results
2.1 Construction of the integrating vector pSET152- EVHB
The 311-bp PermE promoter fragment was amplified with the primer pair P1 and P2 using the genome from S. erythraea as a template. The 603-bp vgb gene was amplified with the primer pair P3 and P4 using the plasmid pRK404-VHb as a template. The 0.9 kb chimeric gene, in which the vgb gene was under the control of the PermE promoter was generated by SOE-PCR with the primer pair P1 and P4 (Figure 1A). DNA sequencing of the chimeric gene showed that the start codon of the vgb gene was successfully changed from ATG to GTG while the second codon was changed from TTA to CTG because of the introduction of the mutation site in primers P2 and P3 (Table 1). The chimeric gene was then cloned into pSET152 to generate the final integrating vector pSET152EVHB (Figure 1C) which has the following fea-tures: site specific reorganization system int/attP encoded by Streptomyces-phage ΦC31, which can be integrated into the attB site of the S. spinosa chromosome; apramycin re-sistance gene aac3(IV); conjugational transfer starting site (oriT); and vgb gene under control of the PermE promoter. Two bands (5.5 and 0.9 kb) were observed by double diges-tion of pSET152EVHB by EcoR I/Xba I (Figure 1B). The fragments accorded with the predicted DNA digestion fragment sizes, which indicated that the integrating vector was successfully constructed.
2.2 PCR and Southern blotting detection of S. spi-nosa S078-1101
PCR was used to check the conjugant S. spinosa S078-1101. A 750 bp fragment of the apramycin resistance gene was obtained with the primer pair Apr-F and Apr-R. A 0.9 kb
Figure 1 Construction of the integrating vector pSET152EVHB. A, The SOE-PCR products. Lane 1, DNA marker; lane 2, PCR product with primer pair P1/P2; lane 3, PCR product with primer pair P3/P4; lane 4, PCR product with primer pair P1/P4. B, The EcoR I/Xba I double digestion product of the
pSET152EVHB vector. Lane 1, DNA marker; lane 2, pSET152EVHB vector digested with EcoR I and Xba I. C, The profile of pSET152EVHB vector.
176 Luo Y S, et al. Sci China Life Sci February (2012) Vol.55 No.2
Table 1 Bacterial strains, plasmids and primers used in this study
Wild-type erythromycin-producing strain, containing erythromycin resistance gene promot-er PermE in its chromosome
NRRL2338
S. spinosa SP06081 Wild-type spinosad-producing strain isolated by our Lab CCTCCNO: M208034
S. spinosa S078-1101 ApraR and vgb integrated to the chromosome of SP06081 at attB site This work CCTCCNO: M2011400
Plasmids
pMD18-T vector AmpR, cloning vector TaKaRa
pRK404-VHb pRK404 harboring whole VHB gene, TetR China Agricultural Univer-sity Prof. Wen Ying [10]
pSET152 ApraR, ΦC31-derived integration vector, pUC18 replicons Huazhong Agricultural University Prof. Tao MeiFeng [34]
pSET152EVHB vgb under the control of PermE promoter inserted into pSET152 by EcoR I and Xba I This work
Primers
P1 GAATTC GGT ACC AGC CCG ACC CGA GCA C This work
P2 GAT GTT AAT GGT TTG CTG GTC CAGCAC CGC TGG ATC CTA CCA P1/P2 were used in PermE promoter PCR amplification (the enzyme restriction site in primer P1 is shown with italic, the underlined bases in primer P2 indicate the mutant sites)
This work
P3 TGG TAG GAT CCA GCG GTGCTG GAC CAG CAA ACC ATT AAC ATC This work
P4 TCTAGA ATG CCA AGG CAC ACC TGA AG P3/P4 were used in vgb gene PCR amplification (the enzyme restriction site in primer P4 is shown with italic, the underlined bases in primer P3 indicate the mutant sites)
This work
Amr-F GTCCAATACGAATGGCGAAAAGC This work
Amr-R ATAACATTCTTCGCATCCCGCC Apr-F/Apr-R were used in PCR amplification of the apramycin resistant gene aac3(IV)
This work
Figure 2 PCR confirmation of the transconjugant S. spinosa S078-1101. M, 200 bp DNA ladder; lanes 13, PCR product of apramycin resistance gene amplified with primer pair Apr-F/Apr-R; lanes 46, PCR product of vgb gene amplified with primer pair P1/P4; lanes 1 and 4, negative control using SP06081 genome as template for PCR; lanes 2 and 5, positive con-trol using plasmid pSET152EVHB as template for PCR; lanes 3 and 6, the
S078-1101 genome was used as a template.
chimeric gene containing the vgb gene and PermE promoter was obtained with the primer pair P1 and P4 (Figure 2) and then confirmed by DNA sequencing. It suggested that the vgb gene under the control of PermE promoter was integrated into the chromosome of SP06081 together with apramycin resistance gene. When using digoxigenin labeled vgb as a probe, Southern blotting showed that the S078-1101 strain had a 3.2 kb genomic size hybridization signal, which was absent in the wild type SP06081 strain (Figure 3A and B), and that the fragment size accorded with the predicted di-
gestion fragment (Figure 3C). All the results demonstrated that the recombinants S. spinosa S078-1101 was success-fully constructed.
2.3 Detection of biological activity of VHb in the re-combinant S. spinosa
A carbon monoxide differential spectrum assay was per-formed using the cell coarse extracting liquid of the S. spi-nosa S078-1101 after 5 d fermentation. The results showed strong absorption peaks at 420 nm from the sample of the S078-1101 strain while it was absent in the control sample of the SP06081 strain (Figure 4). This suggested that VHb was expressed in the recombinant strain S078-1101 and could absorb CO to form carboxyhemoglobin, and thereby was biologically active.
2.4 Effects of VHb expression on the growth and spi-nosad biosynthesis in S. spinosa
Compared with the original SP06081 strain, the S078-1101 strain grew more slowly in CSM. After inoculation, the SP06081 strain reached the exponential phase in 24 h and the stationary phase in 84 h, while the S078-1101 strain reached the exponential phase in 48 h and the stationary
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Figure 3 Southern blotting analysis of the transconjugant S. spinosa S078-1101. A, DNA agarose gel electrophoresis before Southern blotting membrane transfer. B, Hybridization of genomic DNA using digoxigen-in-labeled vgb probe after Southern blotting membrane transfer. M, DL15000 DNA marker; lanes 1 and 2, S. spinosa SP06081 and S078-1101 genomic fragment digested by Pst I respectively, sample loading amount was 10 μg; lane 3, plasmid pSET152EVHB as a positive control, sample loading amount was 0.5 ng. C, The physical maps of pSET152EVHB in-
serted into the S. spinosa SP06081 chromosome at the attB site.
phase hour in 108 h. The growth rate of the S078-1101 strain during exponential phase was lower than that of the SP06081 strain. However, the difference in growth between the two strains tended to become smaller after the stationary phase, and the cell density of the S078-1101 strain appeared slightly lower than that of the SP06081 strain after 120 h culture (Figure 5A). Both strains reached maximum bio- mass after 9 d culture in fermentation medium. The biomass of the S078-1101 strain was slightly lower than that of the
Figure 4 Carbon monoxide differential spectrum assay of VHb. A, S. spinosa SP06081. B, S. spinosa S078-1101.
SP06081 strain during the 5–11 d fermentation, but the dif- ference was not significant (P>0.05) (Figure 5B). These results indicated that integration of the vgb gene into the chromosome of S. spinosa inhibited its growth to some ex-tent.
The spinosad yields of the two strains during the fer-mentation appeared to vary in a similar manner (Figure 5C). Spinosad production was first detected after 5 d fermenta-tion. Subsequently, it increased sharply and reached maxi-mum yield after 9 d fermentation, and then decreased. The spinosad yields of the S078-1101 strain were significantly higher than the original strain at 7 d after fermentation, and the difference in spinosad production between the two strains became significant with the increase of fermentation time (P<0.01). Our present data demonstrated that VHb expression in S. spinosa significantly promoted spinosad biosynthesis.
2.5 Spinosad yield analysis of the recombinant strain under different dissolved oxygen conditions
The effects of vgb expression in S. spinosa on spinosad bi-
Figure 5 Effects of VHb expression on the growth and spinosad biosynthesis of S. spinosa. A, The growth curve of the S. spinosa SP0681 and S078-1101 cultured in CSM. B, The biomass yields of S. spinosa SP06081 and S078-1101 during fermentation. C, The spinosad yields of S. spinosa SP06081 and
S078-1101 during fermentation.
178 Luo Y S, et al. Sci China Life Sci February (2012) Vol.55 No.2
osynthesis were investigated under different dissolved oxy-gen conditions, which were simulated by using shake-flask fermentation with different media quantum at different rota-tion speeds (Table 2). The results showed that the level of dissolved oxygen had a significant influence on spinosad biosynthesis, as the spinosad yields of SP06081 and the recombinant strains decreased markedly with the increase in media quantum or a decrease in rotation speed. Compared with normal oxygen supply conditions, the spinosad yields of the two strains were reduced to more than 50% under moderate oxygen-limiting supply conditions. However, the spinosad yields of the recombinant strain were significantly increased whether at normal oxygen levels or moderate ox-ygen-limiting conditions compared with that of the original strain (P<0.01). Moreover, the spinosad yields of the two strains under highly oxygen-limiting conditions decreased more than 97%. There was no significant difference be-tween the two strains (P>0.05) in spinosad biosynthesis although the yields were higher in the recombinant strain compared with the SP06081 strain.
2.6 Stability of the recombinant strain of S. spinosa
The proportion of cells showing apramycin resistance was determined in cultures grown in the absence of apramycin to investigate the stability of the S078-1101 strain. After five passages, the fraction of colonies which remained apramycin resistant after 5–7 d cultivation in R6 medium without apramycin was greater than 98% (data not shown). This suggested the vgb expression cassette was stably inte-grated into the chromosome of S. spinosa. Further shake-flask fermentation of the different passages with no antibiotic resistance showed that their relative potency of spinosad production was stable and more than 95%. These results demonstrated that the recombinant strain with chro-mosomally integrated vgb gene was genetically stable.
3 Discussion
Although the biosynthetic gene cluster and pathway of spi-nosad have been elucidated, little is known about the regu-lation of these genes’ expression. It is difficult to improve secondary metabolite production by orienting genetic ma-nipulation of S. spinosa. In our previous studies, we iso-
lated a spinosad-producing S. spinosa SP06081 strain, but despite its stable heredity, the spinosad yield of SP06081 was lower and the dissolved oxygen supply was too de-manding for the fermentative production of spinosad. The positive correlation between the potency against hypoxia and spinosad production was also observed during the tradi-tional genetic improvement of the SP06081 strain. Moreo-ver, differential proteomic analysis of S. spinosa SP06081 and the spinosad-hyperproducing PR2 strain, which was obtained from its protoplast regeneration, demonstrated that a group of proteins involved in anti-oxidant stress was sig-nificantly up-regulated in the PR2 strain [37]. For the aforementioned reasons, further investigations on the im-provement of oxygen uptake properties of the SP06081 strain were conducted to promote spinosad biosynthesis. Heterologous expression of VHb has been reported to im-prove cell growth, protein synthesis, metabolite productivity and nitric oxide detoxification. The strategies applied to vgb gene expression include site-specific integration into the chromosome [1921], co-expression [14,15,22] and fusion expression [17]. Our original attempt was to express VHb in S. spinosa by a replicable vector pJN100-VHb which was constructed by cloning vgb ORF into the E. coli/Streptomyces shuttle vector pJN100 [38]. Unfortunately, no positive clones were obtained when pJN100-VHb was transformed into the SP06081 strain, whether by electro-poration of mycelia, PEG-mediated protoplast transfor-mation or by conjugation. This may be attributed to the strong host restriction of S. spinosa SP06081, which blocked foreign plasmid duplication. Previous research has indicated there are two bacteriophage ΦC31 attB sites in the chromosome of S. spinosa and that the integration of small fragment DNA at either of the ΦC31 attB sites has no in-fluence on spinosad biosynthesis [32]. Thus, the vgb gene was cloned into the ΦC31-derived plasmid pSET152 [34], yielding the integrating vector pSET152EVHB, which was then introduced into S. spinosa SP06081 by conjugation and integrated into its chromosomes. Despite the lower efficien-cies (approximately 107 per recipient) and DNA fragment deletion for conjugative gene transfer in S. spinosa, a posi-tive conjugant named S. spinosa S078-1101 was obtained, showing genetic stability and expressing VHb with biologi-cal activity.
The biomass of the recombinant S078-1101 strain
Table 2 Effects of the expression of VHb under different oxygen supply conditions on the spinosad biosynthesis of S. spinosa
Normal oxygen 300 mL/20 mL/300 r min1 251.1±4.4a) 466.6±15.2a),b)
Moderately oxygen-limiting 300 mL/20 mL/240 r min1 152.4±6.5a),b) 211.0±2.8a),b)
Highly oxygen-limiting 300 mL/50 mL/240 r min1 5.3±1.9b) 12.9±2.5b)
a) Compared with SP06081 strain under highly oxygen-limiting supply, P<0.01. b) Compared with SP06081 strain under normal oxygen supply, P<0.01. Spinosad yield is expressed as the mean±SD.
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whether in seed medium or fermentation medium was lower than that of the original strain (Figure 5A and B). However, the spinosad yields of the S078-1101 strain were signifi-cantly increased under both normal oxygen and moderate oxygen-limiting conditions compared with that of the SP06081 strain (P<0.01; Figure 5C, Table 2). A similar effect of vgb gene expression was previously demonstrated by Brünker et al. [39]. However, our findings were different to those obtained in Streptomyces lincolnensis by Yang et al. [19]. The expression of VHb in S. lincolnensis had no sig-nificant influence on the growth and antibiotic production under normal dissolved oxygen conditions, and it promoted an increase in growth and lincomycin yield only under ox-ygen-limiting conditions. These results are probably due to the different regulation mechanisms in diverse microorgan-isms under different growth conditions. In addition, no sig-nificant differences in biomass between the original strain and the recombinant strain were observed during fermenta-tion under normal oxygen supply conditions, whereas spi-nosad yields of the recombinant strain notably increased. Microscopic examination also showed no obvious differ-ences in mycelial fragmentation between the two strains. Therefore, the increase of spinosad yield in the S078-1101 strain was probably due to VHb expression, which may im-prove oxygen utilization by cells, thereby accelerating cer-tain oxygen-requiring rate-limiting steps involved in spi-nosad production. This would lead to more metabolic flux towards the spinosad biosynthetic pathway. Further investi-gations are required to elucidate the precise mechanisms of VHb action and regulation in S. spinosa.
The native promoter of vgb (Pvgb) is regulated by dis-solved oxygen (DO) concentration in Vitreoscilla and E. coli. It is induced at low DO levels (less than 2% of air sat-uration) to express soluble hemoglobin, which enables cells to adapt to hypoxic environments. Although Pvgb is func-tional without the need of expensive inducers, it is not di-rectly regulated by dissolved oxygen. Transcriptional activ-ity of the Pvgb in E. coli is modulated by two oxygen sensors, ferredoxin-NADP reductase and aerobic respiratory control A protein [40,41]. As Pvgb is not functional in most gram-positive bacteria or eukaryotic organisms, various promoter strategies have been adopted by researchers to induce expression of VHb in heterologous hosts. Suthar et al. [18] used an α-amylase promoter PAMY1 in Schwannio-myces occidentalis; Horng et al. [22] applied PBAD in E. coli; Feng et al. [20] used Pluxs in Bacillus thuringiensis; and Xiong et al. [16] employed Pdsz in Rhodococcus erythropo-lis. In this study, the first and second codons of vgb were mutated to GTG and CTG respectively by site-directed mu-tagenesis and SOE-PCR. In addition, the vgb ORF was placed under control of the PermE promoter, which has been successfully applied to actinomycetic metabolic engineering [19,42]. Using this strategy, active VHb was successfully expressed in S. spinosa (Figure 4).
To summarize, the recombinant S078-1101 strain has the
following advantages. First, vgb was controlled by a consti-tutively expressed promoter, thus its expression does not need to be induced. Second, the expression of vgb signifi-cantly promoted spinosad biosynthesis in normal oxygen and moderate oxygen-limiting conditions. Third, vgb was stably integrated into the chromosome of S. spinosa, and no significant changes in spinosad production potency were observed after five generations of propagation without anti-biotic selection. These results suggest that integrating vgb in S. spinosa is an effective means of orientation genetic im-provement, by which, the oxygen absorption capacity of S. spinosa can be improved to promote spinosad biosynthesis. Moreover, to exploit the potential of spinosad production to a greater degree, our research directions in the future are as follows: (i) integration of vgb into chromosomes of other S. spinosa with higher capabilities of spinosad production; and (ii) searching for other strong constitutively expressed pro-moters which are functional in S. spinosa.
We thank M. J. Bibb from the John Innes Centre, Prof. Tao MeiFeng from Huazhong Agricultural University and Prof. Wen Ying from China Agri-cultural University for providing some of the vectors and strains used for gene manipulation in this work. This work was supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605), the National High Technology Research and Develop-ment Project of China (Grant No. 2011AA10A203), and the National Nat-ural Science Foundation of China (Grant No. 31070006).
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